Mesenchymal stem cells (MSCs) are strongly immunosuppressive in vivo and in vitro in both animals and humans. Using cloned MSCs from mouse bone marrow, we have shown that MSCs potently inhibit TCR-activated proliferation and cytokine production of freshly-isolated splenocytes. In vivo, MSCs prevented the rejection of allogeneic skin transplants, suppressed antigen-specific DTH responses, and inhibited graft-versus-host disease (GvHD) in mice. Interestingly, MSCs did not affect the IL-2-driven proliferation of T cell blasts, which do not produce inflammatory cytokines unless re-activated. The immunosuppressive effect of MSCs requires the co-presence of IFN3 and another cytokine - either TNF1, IL-11 or IL-12. Such cytokine pairs provoked MSCs to express inducible nitric oxide synthase (iNOS), produce NO, and secrete of large amounts of T cell specific-chemokines, which complement the activity of NO: in co-cultures, these chemokines drove T cells to migrate into proximity with MSCs, where high levels of NO can suppress T cells. Blockade of NO production or chemokine receptors reversed the inhibition of T cells. We recently found that while human MSCs are equally effective in suppressing T cells and have a similar spectrum of chemokine production, they utilize IDO to affect immunosuppression. Thus, we hypothesize that proinflammatory cytokines induce MSCs to produce chemokines and NO (in mouse) or IDO (in human) which act in concert to mediate immunosuppression. We propose two specific aims to prove this hypothesis using mouse and human MSCs to complement each other. We will first investigate the role of chemokines and NO or IDO in mediating immunosuppression of MSCs in both mouse cells and human cells in vitro and in the mouse GvHD model in vivo. Next, we will determine the molecular mechanisms of the regulation of IDO and NO in mouse and human MSC. Since MSC-mediated immunosuppression occurs through inflammatory cytokine-upregulation of iNOS/IDO and chemokines, a better understanding of the mechanisms underlying these effects will lead to better clinical protocols for immune disorders, cancer immunotherapy and vaccine design.